The bulk degradation-recycling program named autophagy plays key roles in maintenance of cellular homeostasis. The hallmark of autophagy is de novo formation of double membrane-bound vesicles called autophagosomes. During this process, the membrane precursors called phagophores capture aberrant cytoplasmic materials, such as protein aggregates, dysfunctional organelles, and intracellular bacteria, and sequester them into the vesicles upon sealing, followed by the vesicle's fusion with lysosomes for deposition of the engulfed contents for degradation. Such a highly unusual, complex process of intracellular membranes requires two specialized ubiquitin-like proteins (Ubls), Atg12 and Atg8. These Ubls form protein-protein and protein-lipid conjugates, respectively, that are crucial for properly scaffolding membranes and recruiting protein factors and cargo materials. Research in this proposal is directed toward understanding these key processes of autophagy through elucidation of the structure and functions of the Ubl conjugates at an atomic scale. In Aim 1, we will explore the mechanisms underlying the lipid conjugation of Atg8. We have found that Atg8 and Atg3 interact with each other through a previously undescribed non-covalent interface. Strategies based on biochemical and enzyme kinetic analyses will be utilized to demonstrate the importance of this interaction and provide new insights into differences and similarities of th Ubl~E2 activation mechanism and canonical ubiquitin conjugation. In Aim 2, we will explore the role of the covalent linkage of the Atg12~Atg5 protein- protein conjugate. The Atg12~Atg5 conjugate is known to promote Atg8 lipidation by stimulating the transfer of Atg8 from Atg3 to PE. Our previous structural studies have established that the covalent conjugation of Atg12~Atg5 creates a novel E3 platform through stabilization of the non-covalent interface between Atg12 and Atg5. However, how exactly this platform achieves E3-ligase like activity remains unknown. Our new strategy of generating an enzyme-substrate intermediate will facilitate crystallographic studies, which ultimately will unveil the role of Atg12~Atg5 in Atg8 lipidation. In Aim 3, we will explore the mechanisms underlying the assembly of the autophagic membrane scaffold, which consists of Atg8 and Atg12 conjugates. New structural insights into how phagophores are stabilized by these Ubls are expected to emerge. Collectively, the information that will be obtained from these aims will generate an integrated understanding of the role of autophagic Ubls. Structural information will be particularly useful for rational development of new inhibitors of autophagy targeting cancer.